The epithelial sodium channel/degenerin (ENaC/Deg) family of ion channels is constituted of proteins that are implicated in mechanosensation, pain sensation, regulation of extracellular fluid volume and airway surface liquid volume. ENaC/Deg channels are gated by diverse stimuli including neuropeptides, mechanical forces, extracellular protons, and some of them are constitutively active, such as ENaCs. These channels are likely organized as homo- or hetero- trimers and are composed of subunits that have a similar topology with two transmembrane domains (TMs) connected by a large extracellular domain with short intracellular N- and C- termini. The first high resolution structure of the extracellular and membrane-spanning domains of an ENaC/Deg channel, Gallus gallus (chicken) acid-sensing ion channel 1 (cASIC1), was recently reported. ASICs are proton-gated channels expressed throughout neurons of mammalian central and peripheral nervous systems that exist in a resting, functionally inactive state, but undergo a rapid activation and desensitization following extracellular acidification. cASIC1 has a chalice-like shape with a large extracellular domain protruding from the plane of the membrane. The extracellular region is organized in discrete subdomains named the palm, knuckle, 2-ball, finger and thumb. The transition between the TMs and extracellular domain constitute the wrist. While the crystal structure provides great molecular details of cASIC1 in the desensitized state, the mechanism of gating of ASICs and other ENaC/Deg channels remains largely undefined. The first specific aim of this proposal will define conformational rearrangements in the wrist and pore of mouse ASIC1a associated with activation and desensitization. In the second specific aim we will address questions regarding the role of the thumb and palm subdomains in activation, desensitization and recovery from desensitization. Conformational changes that occur in restricted areas of the channel following extracellular acidification will be investigated by voltage clamp fluorometry, a technique that provides information regarding local protein motion associated with specific gating steps. The substituted- cysteine-accessibility method will be used to characterize the structure of specific regions in the closed and desensitized states. Studies proposed in this application address fundamental questions regarding the underlying mechanism of gating and desensitization of mouse ASIC1a. Information derived from this work will be valuable to comprehend the molecular mechanisms of regulation of Deg/ENaC channels.